Genes ingenuity pathway analysis unveils smoothelin‐like 1 (SMTNL1) as a key regulatory protein involved in sodium pentobarbital‐induced growth inhibition in breast cancer

Abstract We previously reported that sodium pentobarbital inhibited the growth of the breast cancer associated with the normalization of microcirculatory hemodynamics and oxygenation. Here, we aimed to screen the key regulatory proteins involved in pentobarbital‐induced normalization of microcirculatory hemodynamics in the breast cancer tissues. A nude mice model of xenograft was established using triple negative breast cancer cell line MDA‐MB‐231. After tumor cell implantation, the mice were subcutaneously injected with 50 mg/kg/day of sodium pentobarbital or an equal volume of solvent adjacent to the tumor for 14 days. Liquid chromatography linked to tandem mass spectrometry (LC–MS/MS) was used to analyze the difference in protein expression profile between the two groups. Ingenuity pathway analysis (IPA) was used to perform the canonical pathway analysis, upstream regulators analysis, and protein–protein interaction networks analysis. Screened proteins were confirmed by real‐time quantitative polymerase chain reaction (RT–qPCR) and Western blot analysis. A total of 101 differentially expressed proteins were revealed between groups. Canonical pathway analysis suggested that acute phase response signaling (z = 1, p = .00208), dilated cardiomyopathy signaling pathway (z = −2, p = .00671), and ILK signaling (z = 1, p = .0172) were key pathways with highlight associations. The mRNA and protein expressions of SMTNL1 were found significantly decreased in pentobarbital‐treated tumor tissues compared with those in controls (both p < .01). Nine important protein–protein interaction networks were identified, and of which, two contained multiple downstream regulatory proteins of SMTNL1. In conclusion, SMTNL1 is revealed as a key protein involved in pentobarbital‐induced growth inhibition signaling in breast cancer. SMTNL1 may become a new potential target for tumor microcirculation research.


| INTRODUC TI ON
Breast cancer is the most common cancer accounting for more than 15% of new cancer cases in Chinese women and contributes to the most cancer deaths in the age group of 15-44 years. 1 Adjuvant and neoadjuvant treatments have been proven to reduce the recurrence and mortality of breast cancer but may increase deaths from other causes including heart disease, leukemia, or other second cancers. 2 Especially, triple-negative breast cancer (TNBC), a highly heterogeneous disease, possesses a high incidence of metastases and poor prognosis. 3Multiple therapeutic regimens including poly ADPribose polymerase inhibitors, immune-checkpoint inhibitors, and antibody-drug conjugates have been established in recent years. 4wever, chemotherapy resistance is more frequent to the established therapies in TNBC, and multiple chemotherapy response biomarkers are identified recently. 5Therefore, new optional drugs with potential anti-TNBC effects are urgent to be explored.
Sodium pentobarbital is a commonly used anesthetic agent in animal experiments via its short-acting sedative and respiration depressive effects. 6,7Unexpectedly, phenobarbital shows an inhibitory effect on glioma development in an inducible rat model of neurogenic tumors. 8Xie et al. 9 revealed that signaling pathways including ERK, c-Jun MAPK, and PI3K/Akt were involved in the suppression of in vitro proliferation and migration of glioma cells by pentobarbital.
Pentobarbital is also found to inhibit colon cancer growth and the incidence of liver metastases in a cancer cell-injected mice model. 10nograft mouse models and genetically engineered models are commonly used to investigate pathological mechanisms of the breast cancer. 11Recently, we reported that pentobarbital inhibited breast cancer growth in mice bearing subcutaneous xenograft. 12is inhibitory effect is associated with the ability of pentobarbital to normalize the microcirculatory hemodynamics and oxygenation in tumor tissues.The perfusion of the capillary network is controlled by the vasomotor response of the arterioles. 13To unveil the key proteins regulating the microcirculatory hemodynamics involved in the antitumor effects of pentobarbital, we compared the differentially expressed proteins between pentobarbital-treated tumor tissues and control tumor tissues and screened the key regulatory proteins using Ingenuity pathway analysis (IPA).

| Cell culture
The MDA-MB-231 breast cancer cell line was purchased from the National Infrastructure of Cell Line Resource (NICR), Chinese Academy of Medical Sciences & Peking Union Medical College (China).The cells were then cultured in the Dulbecco's modified Eagle's medium (Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc.).Cell culture was performed in a humidified incubator with 5% CO 2 and maintained at 37°C.The volume of the tumors was calculated as the following formula: Volume = (length × width 2 )/2.

| Protein digestion
Tumor tissues from each mouse were homogenized in the lysis buffer (8 M urea, 50 mM Tris, pH = 8.0).The proteins were extracted and then reduced with dithiothreitol (10 mM) and alkylated with iodoacetamide (55 mM).The proteins were transferred to an ultracentrifugal tube (10 KD) and buffered with 20 mM Tris HCl.Proteins were then digested using trypsin overnight at 37°C.The peptides were de-salted and concentrated using a reversed-phase (C18) cartridge.The solvent in elution products was removed by a vacuum centrifuge.

| Western blot analysis
Western blot analysis of tumor tissues from the treatment group or control group was performed by SDS-PAGE gel and ECL technique for signal detections (Amersham, Arlington Heights, IL).Tissue proteins were electrophoresed and then transferred to a PVDF membrane.The non-specific reactivity was blocked with 5% nonfat dry milk and incubated with primary antibodies at 4°C overnight.

| Analysis results of LC-MS/MS
The tumor samples collected from the sodium pentobarbital-treated group and control group were analyzed by LC-MS/MS, and a total of 4102 proteins were quantified.Of these, 101 differentially expressed proteins were revealed between groups.A Volcano plot indicated that 47 proteins were differentially up-regulated and 54 differentially down-regulated in sodium pentobarbital-treated tumors compared with controls (Figure 2).The top 25 differentially expressed proteins more abundant in sodium pentobarbital-treated or control tumor tissues were shown in Tables 1 and 2, respectively.
The protein accessions, gene symbols, protein descriptions, ratios, and the p-value were summarized.

| Canonical pathway analysis
IPA was used to perform enrichment analysis and investigate the biological significance of the differentially expressed proteins identified between sodium pentobarbital-treated tumors and control tumors.A total of 33 canonical pathways were first identified based on p-value.Among these pathways, acute phase response signaling (z = 1, p = .00208),dilated cardiomyopathy signaling F I G U R E 2 Differentially expressed proteins between sodium pentobarbital-treated tumors and controls.This volcano plot indicates differentially expressed proteins with a ratio ≥ or ≤1.50 in the treatment group than that in the control group.Meanwhile, a p-value <.05 was considered significant.n = 3.

| Upstream regulators analysis
IPA analysis revealed that smoothelin-like 1 (SMTNL1) is an upstream regulator involved in the inhibition of several differentially regulated proteins including FLNC, GAPDH, MYH4, MYL1, and MYOM1 (Table 4).Notably, these proteins are partly consistent with the key proteins in identified canonical pathways dilated cardiomyopathy signaling pathway, and ILK signaling (Table 3).SMTNL1 is a regulatory smooth muscle protein that plays a role in smooth muscle contractility and vascular adaptations related to multiple pathological or physiological processes such as hypertension and pregnancy. 14TNL1 participates in Ca 2+ sensitization regulation by inhibition of myosin light chain phosphatase (MLCP) and actin cytoskeleton polymerization via interaction with tropomyosin. 15SMTNL1 exerts an inhibitory effect on MLCP related to the phosphorylation of MYL9. 16These effects are also modulated by upstream signaling including protein kinase G (PKG) and cyclic guanosine monophosphate (cGMP). 15,17e to its pro-contractive effect on the arteriole smooth muscle, to determine the relationship between SMTNL1 and the anti-cancer effect of pentobarbital, we did RT-qPCR and Western blotting analyses of SMTNL1 and MYL9 using tumor tissues treated with or without pentobarbital.The relative mRNA expressions of SMTNL1 and MYL9 were found significantly decreased in pentobarbital-treated tumor tissues compared with those in controls (both p < .01, Figure 4A).The protein expressions of SMTNL1 and MYL9 were also significantly decreased in pentobarbital-treated tumor tissues compared with those in controls (both p < .01, Figure 4B,C).

| Protein-protein interaction network analysis
IPA analysis was performed on the protein-protein interaction networks.A total of 9 significant interaction networks were identified.The network annotations from the IPA analysis of the 101 TA B L E 1 Differentially expressed proteins are significantly more abundant in sodium pentobarbital-treated tumor issues than in control tumor issues.F I G U R E 3 Canonical pathway analysis.The top three canonical pathways were identified according to significant z-value and p-value.
TA B L E 3 Canonical pathway analysis.

Fold change (Treatment /control) Expected Location Type
Acute phase response signaling (z = 1, p = .00208)with a score of 20 and 13 focus molecules as well (Figure 5B).

| DISCUSS ION
Compared to non-TNBC, TNBC displays distinctive malignant behavior, with increased invasiveness and poor prognosis. 18ltiple anti-angiogenic agents have been used to treat TNBC, but limited efficacy is observed and the potential mechanisms are not completely understood. 19Current evidence suggests that a vessel normalization strategy is beneficial to the treatment of breast cancer. 20To restore the structure and function of tumor vasculature, vessel normalization promotes the perfusion of blood flow and decreases the hypoxia and acidosis in tumor tissues. 21ltiple agents, especially antiangiogenic agents, could normalize the tumor vessels and improve the oxygenation and delivery of drugs or immune cells. 22e microcirculatory state including hemodynamics and oxygenation plays important role in the malignant behavior of TNBC.
We recently revealed that the inhibitory effect of pentobarbital on breast cancer growth is associated with the ability of pentobarbital to normalize the microcirculatory hemodynamics and oxygenation in tumor tissues. 12The dose of pentobarbital (50 mg/kg/day) used in vivo in our previous and current studies is the routine dose as an anesthetic agent in mice experiments.Due to the fact that the blood flow perfusion is regulated by vascular tone controlled by smooth muscle cells in the resistance vasculatures, 14 we hypothesized that key proteins regulating smooth muscle contractility are potentially involved in the antitumor effect of sodium pentobarbital.
Furthermore, a comparison of the differentially expressed proteins in breast cancer treated with or without pentobarbital could identify key regulatory proteins in the microcirculatory hemodynamics of tumor tissues.This is a pilot study to determine if this method could be used to reveal new potential targets for tumor microcirculation research.
The current study revealed 101 differentially expressed proteins between groups using LC-MS/MS.We then used the IPA software to analyze the biological functions of the 47 differentially up-regulated and 54 differentially down-regulated proteins from pentobarbital-treated samples.IPA is a web analytical tool that provides meaningful huge protein data on comprehensive regulatory pathways and functional networks. 23Firstly, we identified three canonical pathways with highlight associations, including acute phase response signaling, dilated cardiomyopathy signaling pathway, and ILK signaling.The proteins associated with microcirculatory regulation gain more attention in the signaling pathways.
Notably, the last two pathways share 3 proteins including MYH1, F I G U R E 5 Protein-protein interaction network analysis.Two interaction networks with the key downstream proteins regulated by SMTNL1 are focused.(A) No. 4 network annotation (Table 5) is found to be of developmental disorder, hereditary disorder, organismal injury, and abnormalities with a score of 20 and 13 focus molecules out of the 101 differentially expressed proteins between groups.(B) The top diseases and functions of No. 2 network annotation (Table 5) include organ morphology, organismal injury, and abnormalities, skeletal and muscular disorders, with a score of 20 and 13 focus molecules.n = 3.
Animal care and experimental protocols have been approved by the Institutional Animal Care and Use Committee at the Institute of Microcirculation, Chinese Academy of Medical Sciences (China, Approval No. WXH20210109).Xenograft model was established in eight 6-week-old female BALB/c nude mice, which were purchased from the SPF Biotechnology Co., Ltd.(Beijing, China).The mice were housed for 1 week before the model establishment.The mice were free to regular diet and water at 24 ± 1°C under a 12 h-12 h light/ dark cycle.Cell suspension containing 5 × 10 6 MDA-MB-231 cells in Matrigel (100 μL, BD Biosciences, San Jose, CA) was inoculated into the armpit of the nude mice.The mice were randomly divided into two groups (n = 4 in each group) 4 days after the implantation of the tumor cells.Mice in the treatment group were subcutaneously injected with 50 mg/kg/day of sodium pentobarbital (in 100 μL water, Sigma, St. Louis, MO) adjacent to the tumor for 14 days.Other mice injected with an equal volume of solvent served as controls.
Data-independent acquisition (DIA) was performed following the parameters: Positive mode was used.One cycle contains one full scan with a maximum injection time of 54 ms, and 40 segment fragment scans.Full scan ranged from 350 to 1300 m/z and screened at 120 000 resolutions.Fragment spectra were collected at 3000 resolutions.The raw data of DIA were analyzed with Spectronaut software (version 14.3, Biognosys, Schlieren, Switzerland).Raw files were searched against the SwissProt database.The samples were quantitative evaluation based on the MS2 area.Cross-runs were normalized according to the global abundance area.

2. 5 |
Real-time quantitative polymerase chain reaction (RT-qPCR) Total RNA was collected from the tumor issues by using a Trizol reagent (Invitrogen, Carlsbad, CA).Total RNA at 1 μg was transcribed into cDNA using the HiFiScript cDNA Synthesis Kit (Cwbio, Beijing, China) in a total reaction volume of 15 μL.Then qRT-PCR was performed using SYBR FAST qPCR Master Mix Kit (Kapa Biosystems, Woburn, MA) on an ABI StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA).The sequences of primers were as follows: SMTNL1 forward 5′-TGCCA ATG ACA GAG ACAAGC-3′ and reverse 5′-TTGCA TCA GCC TCC TCTTTT-3′; MYL9 forward 5′-TCTTC GCA ATG TTT GAC CAGT-3′ and reverse 5′-GTTGA AAG CCT CCT TAA ACTCCT-3′; GAPDH forward 5′-ACAAC TTT GGT ATC GTG GAAGG-3′ and reverse 5′-GCCAT CAC GCC ACA GTTTC-3′.Real-time PCR was performed for 3 min at 95°C, followed F I G U R E 1 Sodium pentobarbital inhibits tumor growth in vivo in a mice xenograft model.(A) Schematic of the experimental design.Xenograft model was established in eight 6-week old female BALB/c nude mice, which were housed for 1 week until cell suspension containing 5 × 10 6 MDA-MB-231 cells in 100 μL Matrigel were inoculated into the armpit of the mice.At 4 days after the implantation of the tumor cells, the mice in the treatment group were subcutaneously injected with 50 mg/kg/day of sodium pentobarbital (in 100 μL water) adjacent to the tumor for 14 days.Mice in the control group were injected with an equal volume of water.The tumor issues were used for subsequent analysis.(B) Morphologic characteristics of the xenograft tumors treated with or without sodium pentobarbital.(C) The tumor growth, expressed as the volume, was dynamically measured after treatment with or without sodium pentobarbital.(D) The weight of tumors was significantly decreased after the mice treated with sodium pentobarbital than that in controls.n = 4, *p < .05,**p < .01 vs. Control.by 40 cycles of 3 s at 95°C and 20 s at 60°C.GAPDH served as internal control and each sample was analyzed in triplicates.The relative gene expression was determined by the 2 −ΔΔCt method.

SPSS 21 .
0 version software (SPSS Inc., Chicago, IL) was used for statistical analysis.Data are presented as mean ± standard deviation (SD).Student's t-test was used for a two-group comparison.A p-value <.05 was considered as statistical significance.Proteins differentially expressed in pentobarbital-treated tumor tissues as compared with control tumor tissues were determined by above or below the expression ratio of 1.5 and p ≤ .05were considered significantly elevated or decreased.Then the proteins were investigated using IPA Analysis (Ingenuity Systems, Redwood City, CA).The canonical pathways, upstream regulators, and protein-protein interaction networks were analyzed to determine their biological significance.Fisher's exact test was used to calculate a p-value in IPA analysis to determine the significance.In upstream regulators analysis, overlap p-value and activation z-score were both used to predict the regulatory relationship between upstream regulators and their targeted genes.p-value <.05 and z-score >2 or <2 were considered significant.In canonical pathway analysis, pvalue <.05 and z-score ≠ 0 were considered significant.In network analysis, p-value <.05 was considered significant.